Identification of resistance gene analogs linked to a powdery mildew resistance locus in grapevine

Oligonucleotide primers, designed to conserved regions of nucleotide binding site (NBS) motifs within previously cloned pathogen resistance genes, were used to amplify resistance gene analogs (RGAs) from grapevine. Twenty eight unique grapevine RGA sequences were identified and subdivided into 22 groups on the basis of nucleic acid sequence-identity of approximately 70% or greater. Representatives from each group were used in a bulked segregant analysis strategy to screen for restriction fragment length polymorphisms linked to the powdery mildew resistance locus, Run1, introgressed into Vitis vinifera L. from the wild grape species Muscadinia rotundifolia. Three RGA markers were found to be tightly linked to the Run1 locus. Of these markers, two (GLP1–12 and MHD145) cosegregated with the resistance phenotype in 167 progeny tested, whereas the third marker (MHD98) was mapped to a position 2.4 cM from the Run1 locus. The results demonstrate the usefulness of RGA sequences, when used in combination with bulked segregant analysis, to rapidly generate markers tightly linked to resistance loci in crop species. Received: 2 May 2001 / Accepted: 3 August 2001     

Anchoring of a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Didier Lamoureux and Anne Bernole contributed equally to this work.     

Construction of nested genetic core collections to optimize the exploitation of natural diversity in <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. subsp. <Emphasis Type="Italic">sativa</Emphasis>

Background  

The first high quality draft of the grape genome sequence has just been published. This is a critical step in accessing all the genes of this species and increases the chances of exploiting the natural genetic diversity through association genetics. However, our basic knowledge of the extent of allelic variation within the species is still not sufficient. Towards this goal, we constructed nested genetic core collections (G-cores) to capture the simple sequence repeat (SSR) diversity of the grape cultivated compartment (Vitis vinifera L. subsp. sativa) from the world's largest germplasm collection (Domaine de Vassal, INRA Hérault, France), containing 2262 unique genotypes.     

Differential expression of proteins in response to ceramide-mediated stress signal in colon cancer cells by 2-D gel electrophoresis and MALDI-TOF-MS

Comparative cancer cell proteome analysis is a strategy to study the implication of ceramides in the transmission of stress signals. To better understand the mechanisms by which ceramide regulate some physiological or pathological events and the response to the pharmacological treatment of cancer, we performed a differential analysis of the proteome of HCT-116 (human colon carcinoma) cells in response to these substances. We first established the first 2-dimensional map of the HCT-116 proteome. Then, HCT116 cell proteome treated or not with C6-ceramide have been compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization-mass spectrometry and bioinformatic (genomic databases). 2-DE gel analysis revealed more than fourty proteins that were differentially expressed in control cells and cells treated with ceramide. Among them, we confirmed the differential expression of proteins involved in apoptosis and cell adhesion.     

A reference integrated map for cultivated grapevine (Vitis vinifera L.) from three crosses, based on 283 SSR and 501 SNP-based markers

We have developed an integrated map from five elite cultivars of Vitis vinifera L.; Syrah, Pinot Noir, Grenache, Cabernet Sauvignon and Riesling which are parents of three segregating populations. A new source of markers, SNPs, identified in ESTs and unique BAC-end sequences was added to the available IGGP reference set of SSRs. The complete integrated map comprises 1,134 markers (350 AFLP((R)), 332 BESs, 169 ESTs, 283 SSRs) spanning 1,443 cM over 19 linkage groups and shows a mean distance between neighbouring loci of 1.27 cM. Marker order was mainly conserved between the integrated map and the highly dense Syrah x Pinot Noir consensus map except for few inversions. Moreover, the marker order has been validated through the assembled genome sequence of Pinot Noir. We have also assessed the transferability of SNP-based markers among five V. vinifera varieties, enabling marker validation across different genotypes. This integrated map can serve as a fundamental tool for molecular breeding in V. vinifera and related species and provide a basis for studies of genome organization and evolution in grapevines.     

Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library

Resistance to grapevine powdery mildew is controlled by Run1, a single dominant gene present in the wild grapevine species, Muscadinia rotundifolia, but absent from the cultivated species, Vitis vinifera. Run1 has been introgressed into V. vinifera using a pseudo-backcross strategy, and genetic markers have previously been identified that are linked to the resistance locus. Here we describe the construction of comprehensive genetic and physical maps spanning the resistance locus that will enable future positional cloning of the resistance gene. Physical mapping was performed using a bacterial artificial chromosome (BAC) library constructed using genomic DNA extracted from a resistant V. vinifera individual carrying Run1 within an introgression. BAC contig assembly has enabled 20 new genetic markers to be identified that are closely linked to Run1, and the position of the resistance locus has been refined, locating the gene between the simple sequence repeat (SSR) marker, VMC4f3.1, and the BAC end sequence-derived marker, CB292.294. This region contains two multigene families of resistance gene analogues (RGA). A comparison of physical and genetic mapping data indicates that recombination is severely repressed in the vicinity of Run1, possibly due to divergent sequence contained within the introgressed fragment from M. rotundifolia that carries the Run1 gene.     

A new experimental model of acute osteomyelitis due to methicillin-resistant Staphylococcus aureus in rabbit

Aims: To assess the impact of antibiotic therapy on severe osseous infections, animal models of chronic bacterial infections have been developed; however, these models suffer from many experimental limitations. The aim of this work was to develop a new model system in which high levels of bacteria are obtained within femoral bone marrow and bone tissue, and such infections are maintained for at least 14 days. Methods and Results: Experimental osteomyelitis was induced in 25 New Zealand white rabbits. A 10⁹ CFU ml^?1 suspension of methicillin‐resistant Staphylococcus aureus was injected into the knee after bone trepanation. On day 3, surgical debridement was performed to mimic a surgical procedure. Animals were euthanized 1, 2, 3, 9 and 14 days post‐inoculation to determine the bacterial counts in marrow and bone, and to evaluate the stability of the infection. Inoculated lesions also were assessed for changes in histological parameters on days 3 and 7 post‐inoculation. At days 1, 2, 3, 9 and 14 post‐inoculation, we observed 6·50 ± 0·64, 7·30 ± 0·49, 7·82 ± 0·19, 8·00 ± 1·48 and 8·99 ± 0·20 log10 CFU g^?1 in bone marrow and 8·40 ± 0·68, 7·65 ± 0·27, 7·58 ± 0·30, 8·88 ± 0·52 and 8·28 ± 0·39 log10 CFU g^?1 in bone tissue, respectively. No statistical differences in bacterial count were found between bone marrow and bone tissue at any time point. Conclusion: This new model of acute osteomyelitis was validated by histological and microbiological changes in the absence of sclerosing agents, and these changes remained stable for 14 days. Significance and Impact of the Study: These results describe a new experimental model of acute osteomyelitis and demonstrate its usefulness in assessing the activity of antibacterial agents in vivo soon after bone infection.     

Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. E. Paux and F. Legeai contributed equally to this work.